The cell membrane has a 2-dimensional liquid-like structure containing domains that form and disperse continuously on various time and space scales. Rafts are membrane domains that require lipid interactions for their formation. The long-term objective of this proposal is to better understand the molecular mechanisms by which rafts form, are maintained and disintegrate in biological membranes, in particular in the plasma membrane of fiber cells of the eye lens. Detergent insolubility, which has been used to define rafts biochemically, does not reflect pre-existing structures and organization of the membrane. Furthermore, such an approach is not useful for understanding the size, lifetime and dynamics of the raft-constituent molecules and the raft itself. To address these issues, it is proposed to apply the pulse EPR spin labeling technique "discrimination by oxygen transport (DOT)" for in situ studies of rafts in both model and cell membranes. Since the spin-lattice relaxation time of spin labels is sufficiently long, membrane dynamics can be observed on the time scale 0.1 - 100 mu s. The DOT method permits discrimination of different membrane domains because the collision rate between O2 and the nitroxide moiety of spin labels (oxygen diffusion-concentration product) can be quite different in these domains. Additionally, membrane domains can be characterized by profiles of the oxygen diffusion concentration product in situ without the need for separation. This method is especially suitable for obtaining time-space characteristics of small/transient domains. It is hypothesized that rafts form liquid-ordered domains in the plasma membrane liquid-disordered environment. Membrane lipid composition as well as protein content is expected to modulate raft size and dynamics. The DOT method will be used to test the hypothesis on well-defined model systems in which domain size and the lipid exchange rate will be controlled by membrane lipid composition, selected protein and peptide content, and temperature. Furthermore, it will be used to study domain structure in cell membranes. These studies will include mature and aged fiber cell membranes in which the increased cholesterol/lipid ratio and elevated level of sphingomyelin create conditions favoring the formation of rafts. It is proposed: 1) to detect coexisting liquid-ordered and liquid-disordered domains in membranes containing cholesterol;2) to evaluate the size and stability of the raft domains in model membranes made from raft-forming mixtures;3) to examine how membrane anchored proteins and transmembrane alpha-helical peptides affect the organization and dynamics of these lipid raft domains;and 4) to apply the DOT method to look for raft domains in fiber cell plasma membranes of the eye lens during maturation and aging, as well as membrane models of mature, aged and cataractous lenses. Age-related nuclear cataract is a primary cause of blindness in the elderly in third world countries.